In recent years, there have made breakthroughs in DNA sequencing technology, the development of second generation DNA sequencing technology greatly increases the efficiency of genome sequencing and reduces the sequencing time and cost, thus highly facilitating the research on functional genome. In the case of rice, in 2010 the rice molecular biologists in China finished resequencing of 517 endemic rice variety materials in China by using the second generation sequncing technology, constructed high-density haplotype map (HapMap) of the rice, and conducted association study on 14 important agronomic traits of indica variety by using research method of genome-wide association study (GWAS), thus determined relevant candidate gene locus associated with these agronomic traits and established a set of high throughput genotyping identification methods which are effective and quick, mature and stable, precise and of low cost (Huang et al., Genome-wide association studies of 14 agronomic traits in rice landraces. Nat Genet. 2010, 42: 961-967). Soon afterwards, the number of reseqenced rice varieties increased to 950, more sites related to the regulation of flowering time and grain yield were found by association study (Huang et al., Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm. Nat Genet. 2011). Scientists from Huazhong Agricultural University finished the genome-wide resequencing of Minghui 63 and Zhenshan 97 and the cross segregating populations thereof by Illumina sequncing technology, developed a method for high-throughput population genotyping, and constructed an ultrahigh-density genetic linkage map of the cross segregating populations of Minghui 63 and Zhenshan 97 comprising 270,000 SNP markers in 210 recombinant inbred lines, which exhibits strong efficacy in QTL mapping analysis (Xie et al., Parent-independent genotyping for constructing an ultrahigh-density linkage map based on population sequencing. Proc Natl Acad Sci USA. 2010, 107: 10578-10583; Yu et al., Gains in QTL detection using an ultra-high density SNP map based on population sequencing relative to traditional RFLP/SSR markers. PLoS One. 2011, 6: e17595). Recently, a cooperative agreement was signed among Chinese Academy of Agricutural Sciences, Shenzhen Huada Gene Research Institute and International Rice Research Institute, a reseqencing project on 3000 rice core germplasm resources collected in the world was initiated, indicating a overall development of rice genome-wide molecular breeding. By the end of 2010, more than 600 rice genes had been successfully cloned, most of which are related to regulation of important agronomic traits including yield, quality, biotic stress resistance and abiotic stress resistance, and nutrition utilization efficiency, etc. Those genes have powerful potential in breeding (Jiang et al., Rice functional genomics research: Progress and implications for crop genetic improvement. Biotechnol Adv. 2011, 30: 1059-1070). Until 2012, over 800 rice genes hayed been cloned. Such research results involved in rice functional genome study provide important foundational data for rice molecular breeding.
Molecular marker technologies are impotent tools for molecular breeding. Conventional molecular marker techniques, such as RFLP (Restriction Fragment Length Polymorphism) and SSR (Simple Sequence Repeat), play an important role in the research on functional genome. However, conventional molecular marker techniques have many limitations such as low throughout, low quantity and complicated operation processes, and they do not meet the needs of large scale breeding for commercial purpose. In order to precisely regulate a target gene, efficiently select genetic background and accurately analyze and identify the varieties for breeding, there is a need for developing and utilizing high throughput molecular marker techniques. At present, there are mainly two platforms for high throughput molecular marker techniques, one is based on the second generation sequencing technology, and the other is based on gene chip technology. Molecular marker techniques based on gene chip mainly include: SNP array (McNally et al., Genomewide SNP variation reveals relationships among landraces and modern varieties of rice. Proc Natl Acad Sci USA. 2009, 106: 12273-12278), SFP (Single Feature Polymorphism) (Borevitz et al., Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res. 2003, 13: 513-523), DArT technology (Diversity Array Technology) (Jaccoud et al., Diversity arrays: a solid state technology for sequence information independent genotyping. Nucleic Acids Res. 2001, 29: E25), RAD (Restriction site Associated DNA) marker (Miller et al., RAD marker microarrays enable rapid mapping of zebrafish mutations. Genome Biol. 2007, 8: R105; Miller et al., Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. Genome Res. 2007, 17: 240-248), etc. Since SNP sites have the advantages of wide distribution and easy automated detection; among these array-based genotyping technologies, SNP array is most suitable for large scale of commercialized breeding. Currently, Illumina Infinium MaizeSNP50 chip has been used for identification of germplasm resources and association study in maize (Ganal et al., A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome. PLoS One. 2011, 6: e28334; Cook et al., Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels. Plant physiology. 2011), Affymetrix GeneChip Rice 44K gene chip is used for genetic diversity analysis of rice germplasm resource and genome-wide association study in rice (Zhao et al., Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa. Nat Commun. 2011, 2: 467), and Illumina GoldenGate SNP array with different densities have been used for rice molecular breeding (Zhao et al., Genomic diversity and introgression in O. sativa reveal the impact of domestication and breeding on the rice genome. PLoS One. 2010, 5: e10780; Chen et al., Development and application of a set of breeder-friendly SNP markers for genetic analyses and molecular breeding of rice (Oryza sativa L.). Theor Appl Genet. 2011, 123: 869-879; Thomson et al., High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform. Mol Breeding. 2011: 1-12).
Infinium SNP chip technology from Illumina Inc. is a genome-wide SNP detection platform, which is currently well developed and widely used, wherein the chips produced by laser confocal optical fiber bead chip technology and unique bead array technology can bear a huge number of beads, thereby coupling to a large number of SNP probes. At present, the human SNP chips produced by Illumina Inc. can accommodate several millions of SNP markers (http://www.illumina.com). When the chips are produced, each SNP probe sequence comprising 20-50 deoxynucleotides is coupled to specific beads, wherein the types of the beads depend on the number of the loaded SNPs, from several thousands to above ten millions. Each type of beads are coded and detected by their particular address sequence and SNP probe sequence. Each type of beads are repeated 15-30 times on average on each chip, so as to ensure the success rate and repeatability for each SNP to be detected. Illumina Infinium SNP chips have been widely used in the genome variation research on species including human and mice, etc. In the case of lacking stable and effective high-density genome-wide breeding chips in rice breeding field, the chip design based on Illumina Infinium platform according to the present invention satisfies the needs for large scale of rice breeding.